Endothelium forms a thin lining inside blood vessels that acts a barrier to control the response of the vasculature to inflammatory agonists. Several disorders of the vasculature have been identified as major sources of morbidity and mortality in sickle cell disease (SCD). We hypothesize that increased permeability of the vascular endothelium is a disorder of SCD that has not previously been appreciated. Thus, the long- term goal of this project is to define the pathogenetic mechanisms, acute complications, prevention and treatment of endothelial barrier dysfunction in SCD. Since, SCD affects an estimated 100,000 people in the United States (US), and millions more around the world, our long-term goal may have a significant impact on the global health burden. Currently, the vascular disease process involving hypoxia, free heme and ischemia reperfusion in SCD are not well understood. Each of these triggers (hypoxia, ischemia reperfusion and heme) generates oxidative stress, which is well known to increase endothelial permeability and cause tissue edema. In preliminary studies we found increased endothelial permeability and pulmonary edema in two transgenic models of SCD. We acutely increased systemic hemolytic and oxidative stress by intravenously injecting sickle mice with free heme. This caused alveolar flooding and sudden death in SCD mice, while control mice with sickle trait survived. To understand why the response to excess plasma free hemin was so severe, we examined the lung for expression of the acute phase enzyme heme oxygenase-1 (HO-1), which is well known to protect cells from heme. We discovered that HO-1 expression is not elevated in the lungs of mice and humans with SCD, suggesting that the lung uses another mechanism to neutralize the toxic effects of free heme in SCD. We identified NAD(P)H quinone oxido-reductase 1 (NQO1), which is a multi-functional cytoprotective enzyme that scavenges superoxide. NQO1 expression is regulated by the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2). We found that expression of NQO1 is markedly elevated in the lung endothelium of both mice and humans with SCD, suggesting that this enzyme affords protection against oxidants in SCD. Based on these preliminary studies, we will test the OVERALL HYPOTHESIS that "Oxidative stress in sickle cell disease causes potentially fatal endothelial barrier dysfunction that may be attenuated with vasculoprotective therapy". This overall hypothesis will be tested in three inter-related Specific Aims: [1] Define the peripheral and cellular oxidative burden of SCD and the cognate response in transgenic mice [2] Determine whether Nrf2 is essential for protection of the endothelium in sickle cell disease [3] Determine whether lung endothelial barrier integrity in sickle cell disease is dependent on NQO1 activity PUBLIC HEALTH RELEVANCE: Vascular complications are a major problem in individuals who have sickle cell disease. They are believed to be caused by multiple factors including oxidant stress. This study will use several mouse models to find out if oxidant stress causes blood vessels to become leaky in sickle cell disease. We will then test whether we can block this leakage by feeding mice with a specific drug to activate enzymes that neutralize oxidant stress. If the results of this study are positive it will help develop new therapies to manage patients who have sickle cell disease